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Properties of BK_Ca Channels in Oral Keratinocytes

¹ Institute of Oral Medicine, ² Institute of Molecular Medicine, ³ Institute of Basic Medical Sciences, and ⁴ Department of Physiology, National Cheng Kung University Medical College, No. 1, University Road, Tainan 701, Taiwan;

View larger version (28K): [in this window] [in a new window]   Figure 1. Effects of removal of extracellular Ca2+ and various related compounds on IK in normal human oral keratinocytes. Cells were bathed in normal Tyrode’s solution containing 1.8 mM CaCl2. (A) Superimposed current tracings obtained in the absence and presence of external CaCl2. The cell was held at –40 mV, and the depolarizing pulses from –30 to +70 mV were delivered in 20-mV increments. The pipette solution contained 0.1 mM EGTA. The superimposed current traces shown in Aa are control, and those in Ab were obtained 2 min after cells were exposed to Ca2+-free solution containing 1 mM EGTA. (B) The current-voltage relations of outward currents in the presence and absence of extracellular Ca2+ concentration. • : 1.8 mM Ca2+; : Ca2+-free. Mean ± SEM (n = 6–8). (C) Bar graph showing the effects of various blockers of K+ channels on the relative amplitude of IK in normal human oral keratinocytes. In these experiments, cells were bathed in normal Tyrode’s solution containing 1.8 mM CaCl2, and the depolarizing pulses from –40 to +50 mV were applied. Current amplitude in the control was considered to be 1.0, and the relative amplitudes in the presence of each agent were compared. Pax, paxilline (1 µM); Iber, iberitoxin (200 nM); TEA, tetraethylammonium chloride (10 mM); Apa, apamin (200 nM); 5HD, 5-hydroxydecanoate sodium (10 µM); and Anan, anandamide (10 µM). The number of cells from which the data were taken is shown above the bars. Mean ± SEM. *Significantly different from the control group.

View larger version (16K): [in this window] [in a new window]   Figure 2. Effect of caffeic acid phenethyl ester on IK in normal human oral keratinocytes. (A) Original current tracings obtained when the cell was held at –40 mV and the ramp pulses from –80 to +100 mV with a duration of 1 sec were applied. "a" is the control, "b" was recorded in the presence of caffeic acid phenethyl ester (10 µM), and "c" was obtained after the application of caffeic acid phenethyl ester plus paxilline (1 µM). (B) Concentration-dependent stimulation of caffeic acid phenethyl ester on IK in normal human oral keratinocytes. Each cell was depolarized from –40 to +50 mV. Current amplitude in the presence of 300 µM caffeic acid phenethyl ester was considered to be 100%. The smooth line is the fit of the data with a Hill function as described in MATERIALS & METHODS. The values for EC50, maximally stimulated percentage of IK, and the Hill coefficient were 12.8 ± 1.2 µM, 99 ± 1%, and 1.2 ± 0.1 (n = 5). (C) Bar graph showing the effects of caffeic acid phenethyl ester, cinnamyl-3,4-dihydroxy--cyanocinnamate, nordihydroguaiaretic acid, caffeic acid phenethyl ester plus paxilline, and caffeic acid phenethyl ester plus anandamide on the amplitude of IK. Each cell was depolarized from –40 to +50 mV. Current amplitudes were measured at the end of depolarizing pulses. The amplitude in the control was considered to be 1.0, and the relative amplitudes during exposure to each agent were compared. Mean ± SEM (n = 5–7). CAPE, caffeic acid phenethyl ester (10 µM); CDC, cinnamyl-3,4-dihydroxy--cyanocinnamate (10 µM); NDGA, nordihydroguaiaretic acid (10 µM); Pax, paxilline (1 µM); and Anan, anandamide (10 µM). *Significantly different from the control group. **Significantly different from the group with caffeic acid phenethyl ester alone.

View larger version (20K): [in this window] [in a new window]   Figure 3. The activity of BKCa channels in cell-attached patches from normal human oral keratinocytes. Cells were bathed in 145 mM K+ solution containing 1.8 mM CaCl2, and the holding potential was set at +60 mV. (A) Original current tracings obtained in the absence and presence of ionomycin, squamocin, and squamocin plus paxilline. "a" is the control, "b" and "c" were obtained in the presence of either ionomycin (10 µM) or squamocin (10 µM), respectively, and "d" was recorded after application of squamocin (10 µM) plus paxilline (1 µM). (B) Bar graph showing the effects of ionomycin (10 µM), squamocin (10 µM), and squamocin (10 µM) plus paxilline (1 µM) on BKCa-channel activity. Mean ± SEM (n = 6–8). *Significantly different from control group. **Significantly different from squamocin-alone group.

View larger version (18K): [in this window] [in a new window]   Figure 4. Ca2+- and voltage-dependence of the caffeic acid phenethyl ester-induced increase in BKCa-channel activity in inside-out patches of normal human oral keratinocytes. (A) Original current tracings showing the effect of internal Ca2+ on the probability of channel openings. The potential was held at +60 mV. "a", "b", and "c" were obtained after the application of 0.1, 1, and 10 µM Ca2+, respectively. (B) Bar graph showing the stimulation of BKCa channels by caffeic acid phenethyl ester at different concentrations of internal Ca2+. The different concentrations of Ca2+ in the bath before and during exposure to 10 µM caffeic acid phenethyl ester were applied. Each patch was held at +60 mV. Mean ± SEM (n = 4–6). (C) Voltage dependence of the caffeic acid phenethyl ester- and cinnamyl-3,4-dihydroxy--cyanocinnamate-induced increase in BKCa-channel activity of normal human oral keratinocytes. Inside-out patch recordings were performed, and bath medium contained 0.1 µM Ca2+. Caffeic acid phenethyl ester or cinnamyl-3,4-dihydroxy--cyanocinnamate was applied to the intracellular surface of the detached patch. The open probability at +100 mV in the control was taken as 1.0. The activation curves of BKCa channels in the absence (•) and presence of caffeic acid phenethyl ester (10 µM; ) or cinnamyl-3,4-dihydroxy--cyanocinnamate (10 µM; ) were obtained. Mean ± SEM (n = 4–7).

Journal of Dental Research, Vol. 84, No. 5, 468-473 (2005)
DOI: 10.1177/154405910508400513


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